Tumor necrosis factor alpha activates transcription of the NADPH oxidase organizer 1 (NOXO1) gene and upregulates superoxide production in colon epithelial cells.
NADPH oxidase 1 (Nox1) is a multicomponent enzyme consisting of p22(phox), Nox organizer 1 (NOXO1), Nox1 activator 1, and Rac1. Interleukin-1beta, flagellin, interferon-gamma, and tumor necrosis factor alpha (TNF-alpha) similarly induced Nox1 in a colon cancer cell line (T84), whereas only TNF-alpha fully induced NOXO1 and upregulated superoxide-producing activity by ninefold. This upregulation was canceled by knockdown of NOXO1 with small interfering RNAs. TNF-alpha rapidly phosphorylated p38 mitogen-activated protein kinase and c-Jun N-terminal kinase 1/2, followed by phosphorylation of c-Jun and c-Fos and appearance of an AP-1 binding activity within 30 min. We cloned the 5' flank of the human NOXO1 gene (-3888 to +263 bp), and found that the region between -585 and -452 bp, which contains consensus elements of YY-1, AP-1, and Ets, and the GC-rich region encoding three putative binding sites for SP-1, was crucial for TNF-alpha-dependent promoter activity. Serial mutation analysis of the elements identified an AP-1 binding site (from -561 to -551 bp, agtAAGtcatg) as a crucial element for TNF-alpha-stimulated transcription of the human NOXO1 gene, which was also confirmed by the AP-1 decoy experiments. Thus, TNF-alpha acts as a potent activator of Nox1-based oxidase in colon epithelial cells, suggesting a potential role of this oxidase in inflammation of the colon.
Kuwano
Yuki
Y
Department of Stress Science, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan.
Tominaga
Kumiko
K
Kawahara
Tsukasa
T
Sasaki
Hidekazu
H
Takeo
Keiko
K
Nishida
Kensei
K
Masuda
Kiyoshi
K
Kawai
Tomoko
T
Teshima-Kondo
Shigetada
S
Rokutan
Kazuhito
K
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D015152:Blotting, Northern; D002478:Cells, Cultured; D003106:Colon / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D015151:Immunoblotting; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020218:Response Elements; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions; D013481:Superoxides / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C476419:NOXO1 protein, human; D020033:Protein Isoforms; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D008156:Luciferases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases